The initiation of the immune response involves a complex array of intercellular signals. These signals typically involve soluble cytokines coupled with a number of cell-cell contact dependent signals. The contact dependent events, most notably activation of the T-cell receptor, lend specificity to the response whereas the soluble mediators are generally responsible for maintenance of cell differentiation and proliferation. Tumor Necrosis Factor (TNF) and LT-α are two polypeptides generally recognized for involvement with the initiation of the immune response.
TNF and LT-α are soluble proteins noted originally for their ability to inhibit the growth of tumors. [L. Old, “Tumor Necrosis Factor,” Science, 230:630 (1985)]. Further research demonstrated that both proteins exhibit a wide range of activities. TNF is synthesized in response to various inflammatory insults by a variety of cell types including both hematopoietic and nonhematopoietic cells, while LT-α, in contrast, is made specifically by lymphocytes. The two known TNF receptors do not appear to discriminate between LT-α and TNF. [T. Schall et al., “Molecular Cloning and Expression of a Receptor for Human Tumor Necrosis Factor,” Cell, 61:361-370 (1990); C. Smith et al., “A Receptor for Tumor Necrosis Factor Defines an Unusual Family of Cellular and Viral Proteins,” Science 248:1019 (1990)]. In general, LT-α and TNF display similar spectra of activities in in vitro systems, although LT-α is often less potent. [J. Browning et al., Studies Of The Differing Affects Of Tumor Necrosis Factor And Lymphotoxin On The Growth Of Several Human Tumor Lines,” J. Immunol., 143:1859 (1989)].
TNF appears to play a major role in specific aspects of metabolic control, in the response to endotoxin shock, and in the regulation of hematopoietic cell development. [B. Beutler et al., “The History, Properties, and Biological Effects of Cachectin,” Biochemistry, 27, (1988); M. Akashi et al., “Lymphotoxin: Stimulation And Regulation of Colony Stimulating Factors in Fibroblasts,” Blood, 74:2383 (1989); G. Roodman et al., “Tumor Necrosis Factor-alpha and Hematopoietic Progenitors: Effects Of Tumor Necrosis Factor On The Growth Of Erythroid Progenitors CFU-E And BFU-E And The Hematopoietic Cell Lines k562, HL60, And HEL Cells,” Exp. Hematol., 15:928 (1987)].
Along with IL-1 and IL-6, TNF is also a major mediator of the inflammatory response. [D. Cavender et al., “Endothelial Cell Activation Induced By Tumor Necrosis Factor And Lymphotoxin,” Amer. Jour. Path., 134:551 (1989); R. Cotran et al., “Endothelial Activation Its Role In Inflammatory And Immune Reactions,” in Endothelial Cell Biology, (Plenum Press, Simonescu & Simonescu, eds., 1988) 335]. TNF also appears to be involved in T cell activation under certain conditions. [M. Shalaby et al., “The Involvement Of Human Tumor Necrosis Factors-α And-β In The Mixed Lymphocyte Reaction,” J. Immunol., 141:499 (1988); N. Damle et al., “Distinct Regulatory Effects of IL-4 and TNF-α During CD3-Dependent and CD3-Independent Initiation Of Human T-Cell Activation,” Lymph. Res., 8:85 (1989); G. Ranges et al., “Tumor Necrosis Factor-α As A Proliferative Signal For An IL-2-Dependent T Cell Line: Strict Species Specificity of Action,” Amer. Assoc. Immunol., 142:1203 (1989); G. Ranges et al., “Tumor Necrosis Factor α/Cachectin Is A Growth Factor For Thymocytes,” J. Exp. Med., 167:1472 (1988); P. Scheurich et al., “Immunoregulatory Activity Of Recombinant Human Tumor Necrosis Factor (TNF)-α: Induction Of TNF Receptors On Human T Cells And TNF-α-Mediated Enhancement Of T Cell Responses,” J. Immunol., 138:1786 (1987)].
TNF is produced by several types of cells, including monocytes, fibroblasts, T cells and Natural Killer (NK) cells. [D. Goeddel et al., “Tumor Necrosis Factors: Gene Structure And Biological Activities,” Cold Spring Harbor Symposium Quant. Biol., 51, 597 (1986); D. Spriggs et al., “Tumor Necrosis Factor Expression In Human Epithelial Tumor Cell Lines,” J. Clin. Invest., 81:455 (1988); M. Turner et al., “Human T cells From Autoimmune and Normal Individuals Can Produce Tumor Necrosis Factor,” Eur. J. Immunol., 17:1807 (1987)]. Investigators have also detected murine and human forms of TNF that are associated with the surface of various cells either as a transmembrane protein or a receptor-bound molecule. [B. Luettig et al., “Evidence For the Existence of Two Forms of Membrane Tumor Necrosis Factor: An Integral Protein and a Molecule Attached To Its Receptor,” J. Immunol., 143:4034 (1989); M. Kriegler et al., “A Novel Form of TNF/Cachectin Is a Cell Surface Cytotoxic Transmembrane Protein: Ramifications For the Complex Physiology of TNF,” Cell, 53, pp. 45-53 (1988); and M. Kinkhabwala et al., “A Novel Addition To the T Cell Repertory,” J. Exp. Med., 171:941-946 (1990)].
LT-α also has many activities, generally similar, but not identical to those of TNF, including tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation. [N. Ruddle and R. Homer, “The Role of Lymphotoxin in Inflammation,” Prog. Allergy, 40:162-182 (1988)]. Both LT-α and TNF are ligands to members of the nerve growth factor (NGF) receptor family. [S. Mallett and A. N. Barclay, “A New Superfamily Of Cell Surface Proteins Related To The Nerve Growth Factor Receptor,” Immunology Today, 12:7:220-223 (1991).]
In contrast to TNF, LT-α secretion appears to be a specific property of only activated T cells and certain B-lymphoblastoid tumors. [N. Paul et al., “Lymphotoxin,” Ann. Rev. Immunol., 6:407 (1988)]. Some researchers have also indicated that a membrane-associated form of LT-α may be expressed on the surface of lymphocytes under certain circumstances [J. Hiserodt, et al., “Identification of Membrane-Associated Lymphotoxin (LT) On Mitogen-Activated Human Lymphocytes Using Heterologous Anti-LT Antisera In Vitro,” Cell. Immunol., 34:326-339 (1977); C. Ware et al., “Mechanisms of Lymphocyte-Mediated Cytotoxicity,” J. Immunol., 126:1927-1933 (1981); U. Anderson et al. J. Immunol. Methods, 123, 233 (1989); Y. Abe et al., Jpn. J. Canc. Res., 82:23 (1991); Y. Abe et al., “Studies of Membrane Associated and Soluble (Secreted) Lymphotoxin In Human Lymphokine-Activated T-Killer Cells In Vitro,” Lymphokine and Cytokine Research, 11, 2:15-121 (1992)].
In recent years genes for both TNF and LT-α have been isolated and cloned, leading to their complete characterization and to the availability of recombinant forms of both proteins. [P. Gray et al., “Cloning and Expression of cDNA For Human Lymphotoxin, A Lymphokine With Tumor Necrosis Activity,” Nature, 312:121-124 (1984); D. Pennica et al., “Human Tumor Necrosis Factor: Precusor Structure, Expression And Homology To Lymphotoxin,” Nature, 312:724 (1984)].
Other “cytokine-like” cell surface proteins including the CD40 protein have recently been shown to share certain similarities with TNF and LT-α. Like TNF and LT-α, the CD40 protein is a ligand to members of the TNF/nerve growth factor (NGF) receptor family. [S. Mallett and N. Barclay, Immunology Today, 12:220-223 (1991)]. The CD40 protein is a 277-amino acid protein expressed on the surface of B lymphocytes, epithelial cells, and some carcinoma cell lines. [R. Armitage et al., Nature, 357:80-82 (1992); T. Farrah and C. Smith, “Emerging Cytokine Family,” Nature, 358:26 (1992)].
We have now identified a novel surface protein, lymphotoxin-β (LT-β) or p33. LT-β has been identified on the surface of several types of lymphocyte cells, including OKT3-stimulated primary T cells, antigen specific IL-2 dependent CTL clones, and a PMA-stimulated human T cell hybridoma II-23.D7. LT-β targets LT-α produced in the cell to the cell membrane where LT-β and LT-α appear as a complex (designated “LT-α/LT-β” throughout this disclosure). The LT-α/LT-β complex is believed to be a novel mechanism for membrane expression of LT-α by activated T-cells. [Androlewicz et al., “Lymphotoxin Is Expressed As a Heteromeric complex With A Distinct 33 kDa Glycoprotein On The Surface Of An Activated Human T Cell Hybridoma,” Journal Of Biological Chemistry, 267:2542-2547 (1992)]. The LT-α/LT-β complex may exhibit cytolytic and cell regulatory activity similar to the soluble LT-α, TNF and CD40 proteins. The membrane-associated LT-β complexed with LT-α may represent, as a complex, a novel ligand for T cell interactions with other cells and may also be useful in targeted cell lysis.